Preparation of alginate-based capillary hydrogels

Gel formation: Sodium alginate was dissolved at a concentration of 20 g/L (2% w/w) in purified water. Optionally porcine skin gelatin was added to this solution at a concentration of 2 g/L (0.2% w/w). Electrolyte solutions (Cu(NO₃)₂, Sr(NO₃)₂, Zn(NO₃)₂) were prepared at a concentration of 1 mol/L in purified water. All solutions were subsequently filtered through a Nalgene^® vacuum filtration system equipped with a polyethersulfone membrane with a pore size of 0.2 µm obtained by VWR International (Germany). 65 grams of alginate solution were poured into anodised cylindrical aluminium moulds (5.5 cm in diameter and 4 cm in height), covered by a glass lid, and allowed to stand for about 2 h; the alginate molecules should get close contact with the hydrophilic walls of the mould which is important for the stability during gel formation. Then the electrolyte solution was sprayed onto the alginate solution using a pump spray bottle (VWR) until the alginate was covered by a 5 mm thick layer of electrolyte solution (~10 mL); after several minutes another 10 mL of electrolyte solution were filled onto the alginate using a piston pipette. Moulds were covered by a lid and allowed to stand for at least one day until gel formation was finished. With the help of a spatula the metal alginate or metal gelatin/alginate gels were carefully removed from the aluminium moulds and immersed in sterile filtered water or 2 g/L gelatin solution, respectively, to remove excessive electrolyte; the water or gelatin solution was changed after at least 4 h for not less than 4 times.

Gel cutting: The obtained gel bodies were cut perpendicular to the longitudinal axis of the capillaries using a custom-made cutting machine. Gel bodies were mounted onto a slide, whose position in relation to the blade could be adjusted in a defined way. After removing the non-structured top layer of the gel (5 mm) one slice of 15 mm in thickness was cut from the gel cylinder.

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Figure 3.1: Custom made cutting machine. Machine showing sample holder, cutting blade and moving stage (A), close up view of blade approaching towards sample holder (B), alginate gel in sample holder and blade cutting the gel (C).

Dehydration, crosslinking and ion exchange: Gel slices were dehydrated by equilibrating in acetone/water mixtures of rising acetone content (25%, 50%, 75%) for a minimum of 4 h per step; then the gels were equilibrated in pure acetone and then in dry acetone twice for not less than 4 h per step; solutions are slightly agitated by stirring. Hexamethylene diisocyanate (HDI) was dissolved in dry acetone at a concentration of 0.1 mol/L and dehydrated acetone-soaked alginate gels were immersed in HDI solution for 4 h under slight stirring. Gels were removed from HDI solution and immersed in dry acetone for 5 min to remove HDI from the capillary lumens. Then the gel slices were put between two filter papers and dried in air for 10 min to remove acetone from the capillary lumens. The gels were immersed in sterile filtered water (with a load from the top because gels are swimming on the water interface) for 4 h under slight stirring. Then they were heated in water to 70°C for additional 2 h until carbon dioxide development stopped. After that, the gels were immersed in 1 mol/L hydrochloric acid solution (HCl) five times for at least 2 h under slight stirring to remove the crosslinking divalent cations. Finally, the gels were immersed in sterile filtered water for several times until the water reached a neutral pH. Gels were sterilised by incubation in 70%

ethanol for 5 min and finally kept in sterile phosphate-buffered saline (PBS; pH 7.4).

3.2.1.1 Characterisation of alginate hydrogels

Pore structure: Blocks of alginate hydrogels (5 mm x 5 mm x 15 mm) were cut from the gel cylinders using razor blades. Gel slices with 500 µm in thickness were prepared from these blocks using a vibrating microtome VT1000 S of Leica (Germany). These thin slices were immersed in a 200 mg/L solution of ruthenium red in water for 1 h to enhance the contrast for light microscopy analysis (Figure 3.1). Images of the capillary structure were prepared using

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a Eclipse E-400 light microscope from Nikon (Japan) in 50 to 200-fold magnification, a TK-1380-E Video Camera from JVC (Japan), and the Studio Quick Start software, version 8, from Pinnacle Systems Inc. (USA). Images were analysed by a self-made computer program regarding capillary diameter and capillary density. For each type of gel a minimum of 10 different images was used for structure characterization.

Figure 3.2: Alginate gel slices in ruthenium red solution for pore structure analysis.

Ion exchange: The efficacy of the ion exchange process was controlled by atomic emission spectroscopy. The gel cylinders were cut into slices of 2 mm in thickness using our custom-made cutting machine and then pieces of 10 mm in diameter were prepared using a hollow punch. The ion exchange process was performed 0 – 5 times by immersing the small gel specimens in 1 mol/L HCl solution for a minimum of 2 h per step. At the end, all gel samples were washed with water until a neutral pH was reached and dried by lyophilisation. The weight of each sample was measured using a MT-5 electronic microbalance of Mettler Toledo (Germany). The dried gel samples were individually placed in small porcelain cups and heated at 120°C after addition of 2 ml of ultra-pure concentrated HNO3 until the liquid was completely vaporised. Then 2 ml of ultra-pure concentrated HClO4 were added and the solution was heated again until the liquid was completely vaporised. The decomposition procedure using HClO4 was repeated until a colourless remnant remained. This remnant was carefully dissolved in 0.5 mol/L ultra-pure HNO3 solution and transferred into a calibrated 10-ml glass flask. The solutions obtained after alginate gel decomposition were analysed regarding their metal ion content by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using a Spectroflame-EOP of Spectro Analytical Instruments GmbH

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(Germany). Standard solutions were prepared within the concentration range 0.01 – 100 mg/L. The identified amount of the remaining cation was related to the weight of the dried gel sample (nmol cation / mg dried gel). A minimum of three specimens per type of cation and number of HCl washings was analysed.

Figure 3.3: Alginate gels in the form of beads (A, B) and slices (C, D) before (ion removal) HCl washing (A, C) and after (ion removal) HCl washings (B, D).

Stability testing: Small gel slices of copper alginate with 2 mm in thickness and 10 mm in diameter have been used for stability testing. One group of the samples was crosslinked by HDI reaction, the other was not crosslinked. Copper ions were removed by HCl immersion from both groups and then the gel specimens were individually placed into the wells of 24-well culture plates (Nunc^®) which were obtained from VWR. The samples were incubated in 2 ml of PBS solution at 37°C using a Cytoperm^® incubator from Heraeus (Germany) and after 1, 3, 7, and 14 d the liquid was removed. Alginate dissolved from the gel samples was quantified using the Purpald^®-assay after hydrolysis and oxidation of the carbohydrate moieties with NaIO4 as described previously (Avigad G et al. 1983). For hydrolysis, 0.5 ml of 2 mol/L sodium hydroxide solution was added to 1.5 ml of the alginate containing supernatant, transferred into pressure-resistant Schott^® screw cap glass vials (VWR) and heated at 75°C over night. The cooled hydrolysate was neutralised by adding 165 µl 6 mol/L HCl and mixed with 2.835 ml of a 0.1 mol/l acetic acid/sodium acetate buffer solution (pH 5.4) to give a sample volume of 5 ml. Then, 0.5 ml of this solution were transferred into the well of a 24-well culture plate and incubated after addition of 0.5 ml of a 30 mmol/L solution of NaIO₄ in acetic acid/sodium acetate buffer for 1 h in the dark at 20 °C. A 200 µl aliquot of

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the oxidised alginate solution was transferred into a new well and incubated after addition of 300 µl of a 0.1 mol/L Purpald^® solution in 1 mol/L NaOH for 30 min at 20 °C. Finally, 500 µl of a 0.1 mol/L NaBH₄ solution in 1 mol/L NaOH were added. The absorbance of the resulting solution was measured at 546 nm. As blanks, solutions consisting of NaIO₄, Purpald^®, and NaBH₄ without hydrolyzed alginate were used. A minimum of four gel samples per crosslinking condition and time point was used for stability testing.

Gelatin content: Small beads of copper alginate and copper gelatin/alginate were prepared by dropping 2% alginate solution into 1 mol/l Cu(NO3)2 solution by the use of a burette. Beads were crosslinked with HDI and copper ions were removed with HCl as described above.

Then, the beads were placed in different numbers into 2-mL Eppendorf^® cups and incubated with 1.5 ml of the BCA protein assay working solution for 30 min at 40°C. The presence of the gelatin constituent was monitored by the development of a purple colour which was measured at 562 nm on a Lamda-18 UV/Vis spectrophotometer of Perkin Elmer Corp.

(USA). Optical density was related to the number of beads and the dry weight which was determined after freeze drying.